Based on expression analysis of germ cells we had cloned a novel mArd1 (Arrest Defective 1) homolog, which we named mArd2 that demonstrated testis-specificity and elevated expression in pachytene spermatocytes. The mArd1 protein is known to interact with an auxiliary protein subunit mNAT1 to constitute a functional N-acetyltransferase. Earlier studies in yeasts had identified a diverse role for ARD1 from cell cycle regulation to DNA repair and recombination. We showed that the transcript of mArd2 is preferentially expressed in male meiotic germ cells while the expression of the encoded protein was delayed. By performing in vitro protein pull-down assays and N-acetyltransferase activity assay, we demonstrated that Ard2, like Ard1, could interact with Nat1 and display N-acetyltransferase activity. Our data imply that Ard1 and Ard2 are functionally homologous. The expression of Ard2 may therefore be responsible for the compensation of the loss of X-linked Ard1 starting from meiosis. [unreadable] [unreadable] Ard1 and Ard2 display a grossly different expression pattern in various cell lines and mouse tissues examined. Particularly, the Ard2 gene is expressed in a developmentally regulated manner during spermatogenesis. To elucidate the molecular mechanism of the tissue-specificity of Ard2, an Ard2 promoter assay was first performed in two Ard2 non-expressing cell lines, namely NIH/3T3 and GC-2spd(ts), which represent cells of somatic and early germ-line origin, respectively. The detection of Ard2 promoter activities in both cell types indicates that the transcriptional machinery required for Ard2 expression is intact; the absence of Ard2 expression would thus be a result of other regulatory mechanisms. We hypothesize that DNA methylation could play a role in the regulation of Ard2 expression. By treating both NIH/3T3 and GC-2spd(ts) cells with 5-aza-2-deoxycytidine, a reactivation of Ard2 transcription was observed, thus supports the idea that DNA methylation is responsible for regulating Ard2 transcription. The methylation status of the two CpG islands spanning the proximal promoter and half of the coding region of Ard2 gene was examined and compared by sequencing of bisulfite-treated genomic DNA isolated from various tissues and different stages of male germ cells. Our result showed that the proximal promoter of Ard2 was hypermethylated in mouse tissues that do not express Ard2 but hypomethylated in male germ cells which express the gene. Interestingly, the distal CpG island was found to be hypomethylated in stages of germ cells that display a higher Ard2 expression level, but hypermethylated in non-expressing tissues as well as mitotic germ cells in which Ard2 expression is minimal. Our findings illustrate that the testis-specific expression of Ard2 is epigenetically regulated. The Ard1-Ard2 expression system could be a paradigm for studying epigenetic regulation of mammalian spermatogenic gene expression. [unreadable] [unreadable] Another gene we identified based on expression profiling of male germ cells is mLin28. Lin28 is an evolutionarily conserved RNA-binding protein that is implicated in the correct temporal control of cellular development. Since Lin28 is highly expressed only in mitotic male germ cells in the testis, we hypothesized that Lin28 could act as a RNA chaperone which functions to control the availability of transcripts encoding products important to cell fate decision. In this respect, we use the P19 embryonal carcinoma cells as a model. Our preliminary findings indicate the Lin28 protein can bind to specific sets of RNA transcripts that encode ribosomal proteins, components of protein translation and mRNA processing machineries, among others, in vivo. Gene knockdown experiments are now underway to confirm the role of Lin28 in regulating the synthesis of these target proteins and to study their involvement in the decision of cellular fate.